| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Regular Articles |
B-Dependent Proinflammatory Factors but not of Collagen I in a Rabbit Model of Atherosclerosis
From the Research Laboratory*
and Division of
Cardiology,
Fundación Jiménez
Díaz, Autonoma University, Madrid, and the Division of
Pathology,
Hospital Clínico,
Madrid, Spain
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
B (NF-B) has been implicated in
the control of chemokine transcription and cell proliferation,
we also investigated its activation and localization in the lesion.
Quinapril administration for 28 days caused a down-regulation in
arterial expression of interleukin-8 and monocyte chemoattractant
protein-1 (mRNA and protein). However, collagen I expression
(mRNA and protein) was not modified. PDGF-B expression was reduced in
both the intima and the media. Active NF-B, found in both
macrophages and vascular smooth muscle cells, was also reduced
by quinapril. Nevertheless, no significant changes were noted
in the mild neointima formation, although a certain trend
toward normalization was found in the quinapril-treated group. In
conclusion, our results show that quinapril treatment
attenuates several parameters associated with inflammation within the
atherosclerotic lesions that are controlled by NF-B,
although it has no effect on collagen I expression. Both effects could
contribute to the stabilization of the atherosclerotic
plaque.
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Accumulation of VSMC in the neointima is another feature of atherosclerosis and a consequence of their migration from the media and their proliferation within the lesion. Platelet derived growth factor B (PDGF-B) is a potent inductor of VSMC migration and proliferation. It can be produced by VSMC and macrophages and is overexpressed during experimental vascular damage12,13 and in human lesions.14
Nuclear factor B (NF-B) is a common transcription factor involved
in the regulation of many proinflammatory genes15
including
MCP-1,16
IL-8,17
and PDGF-B,18
as
well as in the control of VSMC proliferation.19
Its
activation (the release of the inhibitory IB subunit and the
translocation of the active p50-p65 heterodimer to the nucleus) can be
induced by different stimuli such as cytokines and oxidative
stress.15
The relevance of this nuclear factor in the
development of inflammatory processes, including atherosclerosis, is
becoming evident.20,21
A high level of activity of the angiotensin converting enzyme (ACE) has been found in the neointima following experimental vascular injury22,23 and in humans up-regulation of ACE expression is associated with enhanced risk of myocardial infarction.24 In animal models, ACE inhibitors diminished neointima formation25 and in humans they reduce the incidence of reinfarction.26 Although several potential mechanisms of these effects have been proposed, including plaque stabilization27 and improvement of endothelial function,28 there is not yet any clear explanation.
In this work we have used an experimental atherosclerosis model in
rabbits to study the effect of the ACE inhibitor quinapril on several
parameters of plaque development and stability such as the presence of
chemokines involved in the recruitment of inflammatory cells (IL-8,
MCP-1), the VSMC mitogen and chemoattractant PDGF-B, and the amount of
collagen I. We also studied the activation and localization of NF-B,
a common modulator of MCP-1, IL-8, and PDGF-B gene expression.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Atherosclerosis was induced according to a previously described technique with minimal modifications.29 Thirty-three white New Zealand male rabbits weighing 3.5–4 kg were anesthetized by intramuscular injection of 5 mg/kg xylazine (Rompun, Bayer AG, Leverkusen, Germany) and 35 mg/kg ketamine (Ketolar, Parke-Davis, Ann Arbor, MI). Local relief from pain was achieved by subcutaneous injection of 2% Lidocaine (Braun, Barcelona, Spain). A prophylactic intramuscular injection of 125 mg/kg of cefazoline (Llorente Laboratories, Madrid, Spain) was administered 30 minutes before the surgical procedure and all techniques were done in sterile conditions. Ten mL of blood were drawn from the ear vein for lipid determination by standard methods and serum ACE activity measurement. Arterial pressure was estimated by a sphygmomanometer in the femoral artery. Proximal bilateral femoral arteriotomies were performed and proximal and distal ligatures were placed to isolate a segment of approximately 2 cm, which was cannulated with a 27-gauge needle. A vent was made by needle puncture and blood was removed by a flush of saline. Endothelial damage was induced by the passage of industrial nitrogen at a rate of 80 mL/minute for 8 minutes. The isolated segments were then flushed again with saline and the ligatures were removed. Hemostasis was achieved by local pressure and the wound was closed with a 4.0 vicryl subcuticular suture. After surgery the animals were placed on a 2% cholesterol, 6% peanut oil diet (Letica, Barcelona, Spain) and maintained for 28 days in individual cages until killed. Four control animals were included in the study for comparison and five animals were killed after 2 days of quinapril consumption to quantify vascular ACE activity.
Two days before the surgical procedure the animals were randomized to quinapril (n = 9) (Accupril, Parke-Davis) or no treatment (n = 15). Quinapril was freshly dissolved every other day in drinking water and given to the rabbits at 1 mg/kg/day. The consumption of water was measured every other day and the weight of the animals was recorded weekly during the experiment to confirm the appropriate administration of the drug. Control animals were fed standard chow with no experimental intervention.
Sample Collection and Measurement of ACE Activity
At the time of sacrifice animals were anesthetized with ketamine/xylazine and 10 mL of blood were drawn from the ear vein for biochemical determinations. Both femoral arteries were exposed and ligatures were placed to isolate the damaged segments and flushed with saline. One of the arteries was removed, the adventitial layer was carefully peeled, and the artery was immediately snap-frozen in liquid nitrogen. The aorta was ligated and flushed with saline and a piece was removed and snap-frozen in liquid nitrogen. The animal was euthanized with an overdose of pentobarbital (Abbot, Madrid, Spain) and the other femoral artery was cannulated and fixed in situ with 100 mL of 4% buffered formaldehyde at a pressure of 100 mm Hg. It was then removed and kept for 24 hours in 4% buffered formaldehyde and afterwards in 70% ethanol until embedded in paraffin.
ACE activity determinations were done in serum and in samples of frozen uninjured artery (aorta) by a spectrophotometric method (Sigma Chemicals, St. Louis, MO). The activity is expressed as relative units per milligram protein of tissue as determined by the BCA method (Pierce, Rockford, IL) or as units per mL in serum.
Immunohistochemistry
Paraffin-embedded arteries were cross-sectioned in 4 µm-thick
pieces at 5-mm intervals from the proximal to the distal end, dewaxed,
and rehydrated. Identification of macrophages was performed using a
monoclonal antibody for rabbit macrophages (RAM11, Dako, Carpinteria,
CA).30
MCP-1 and IL-8 were detected with a polyclonal goat
anti-human MCP-1 antibody and a monoclonal anti-human IL-8 antibody,
respectively (Immugenex Corp., Los Angeles, CA). For collagen I, a
polyclonal anti-human antibody (Southern Biotechnology, Birmingham, AL)
was used, and for VSMC a monoclonal anti-rabbit 
-actin (HHF35; ENZO
Diagnostic, New York, NY). Endogenous peroxidase activity was quenched
by incubating the sections in 3% hydrogen peroxide:methanol (1:1) for
30 minutes. Nonspecific antibody binding was blocked by incubation of
the tissue section for 1 hour in suppressor serum consisting of 6%
goat serum and 4% bovine serum albumin (BSA) in phosphate buffered
saline (PBS), pH 7.0, for HHF35 and anti-IL-8 antibodies, and 6% horse
serum and 4% BSA in PBS, pH 7.0, for anti-MCP-1 and RAM11 antibodies.
For collagen I, the suppressor serum was 2% horse serum and 1% BSA in
PBS. HHF35 was applied directly for 1 hour. Anti-MCP-1 and
anti-collagen I antibodies were applied overnight and RAM11 for 1 hour
in 1% horse serum and 4% BSA in PBS and anti-IL-8 was applied
overnight in 1% goat serum and 4% BSA. As secondary antibody, a goat
anti-mouse IgG HRPO-conjugated (Seralab, Sussex, UK) was used for RAM11
and HHF35. For MCP-1 and collagen I a donkey anti-goat IgG
HRPO-conjugated (The Binding Site, Birmingham, UK) was used and for
IL-8 a goat anti-mouse IgG biotin labeled. Secondary antibodies were
applied for 30 minutes to 1 hour and then sections were stained for 10
minutes at room temperature with 0.05% 3,3'-diaminobenzidine
tetrahydrochloride (Dako) and 0.01% hydrogen peroxide in PBS (RAM11,
HHF35, collagen I and MCP-1) or streptavidin-biotin/alkaline
phosphatase (Dako) for 30 minutes plus fast red substrate for 10
minutes (IL-8). Finally, sections were counterstained with hematoxylin
and mounted in Pertex (Medite, Burgdorf, Germany). In each
experiment, negative controls without the primary antibody or using an
unrelated antibody were included to check for nonspecific staining.
Quantification was assessed by two independent observers and the sections with the maximal lesion in each animal were chosen. The MCP-1 and macrophages-occupied area was quantified automatically as described below, but IL-8- and collagen I-stained areas were assessed with a semiquantitative score due to the diffuse pattern of staining. Computer-assisted morphometric analysis was performed with the Cue-2 semiautomatic image analysis system (Olympus, Hamburg, Germany). The arterial cross-sections stained with antibodies were digitized by an Olympus microscope (BH-2) connected to a CCD video camera. The labeled areas in the intima and the media were delimited and after image enhancement and segmentation (transformation to a binary image) a grey value ranging from 0 to 255 was assigned to each pixel and automatic analysis was performed. Results were expressed as immunostained area and fractional area of intima and media.
The lesion size was measured on orcein-stained arterial sections with the NewSketch 1212 graphic tablet (Genius, Ontario, CA) linked to a microcomputer using the autoCADD 10.0 software (Autodesk AG) as previously described.20
RNA Extraction, Reverse Transcriptase Polymerase Chain Reaction, and Northern Blot
Frozen femoral arteries were pulverized in a metallic chamber. Total RNA was obtained by the acid guanidine-thiocyanate-phenol-chloroform method31 and quantified by absorption at 260 nm in duplicate. Equal amounts of RNA (5 µg) were denatured and electrophoresed in a 1% agarose-formaldehyde gel to check the absence of degradation of RNA. Equal amounts of RNA from each animal of every group (control, untreated, and quinapril-treated) were pooled. For RT-PCR, 1 µg of RNA from every pool was applied and primers based on the published sequences for IL-8, MCP-1,32 and glyceraldehyde-3'-phosphate dehydrogenase (G3PDH)33 were used.
For Northern blot, 30 µg of RNA were denatured and electrophoresed in
a 1% agarose-formaldehyde gel, transferred to nylon membranes
(Genescreen, New England Nuclear, Boston, MA) and carried out as
previously described.31
The probes for collagen
I34
and G3PDH33
were obtained from PCR products
and labeled with -[32P]-dCTP (Amersham,
Buckinghamshire, UK) by the random priming method (Promega,
Madison, WI). G3PDH was used as internal control to calculate
the relative expression of each mRNA. Films were scanned using the
Image Quant densitometer (Molecular Dynamics, Sunnyvale, CA).
In Situ Hybridization
Digoxigenin-labeled single-stranded RNA probe of PDGF-B was prepared using a nonradioactive RNA labeling kit (Boehringer Mannheim, Mannheim, Germany) according to the manufacturer's instructions. Sense and antisense PDGF-B riboprobes were synthesized from the linearized plasmid PCR 3 (Invitrogen, San Diego, CA) containing a PCR fragment (462 bp) of rat PDGF-B35 as run-off transcripts with T3 and T7 RNA polymerases in the presence of digoxigenin-11-UTP.
Paraffin-embedded tissue sections were cut 4 µm thick and floated onto APES (Sigma) coated slides. The tissue sections were heated at 65°C overnight, then fixed with 1.5% paraformaldehyde-1.5% glutaraldehyde. After dewaxing, tissues were incubated in 5 mmol/L levamisole to inhibit endogenous phosphatase. Deproteinization was carried out in 0.2 mol/L HCl followed by digestion with proteinase K and washed twice in 0.2% glycine for 5 minutes. After digestion, all sections were postfixed as above, dehydrated through a graded ethanol series, and air dried at room temperature. The slides were hybridized with 10 ng/mL denatured digoxigenin-11-UTP-labeled riboprobes in hybridization buffer (2x SSC, 1x Denhardt's solution, 0.1 mol/L sodium phosphate, pH 6.5, 10% dextran sulfate in formamide). Sealed cover slips were placed over the tissue sections and hybridization was allowed to occur overnight at 42°C in a moisturized chamber. The cover slips were removed and the slices were washed in 2x SSC for 1 hour at room temperature and 0.2x SSC for 30 minutes at 37°C. The sections were incubated with an antidigoxigenin antibody conjugated with alkaline phosphatase (Boehringer Mannheim) for 30 minutes at 37°C. Colorimetric detection of RNA-RNA hybrids was performed with nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolylphosphate (X-phosphate) in the dark for 1–4 hours. The color reaction was stopped with 10 mmol/L Tris-HCl, 1 mmol/L EDTA, pH 8.0, and cover slips applied with 60% glycerol before microscopic examination. The negative controls included hybridization with the sense probe, RNase treatment before hybridization, and omission of the RNA probe. Semiquantitative scores were assigned.
NF-B Determination by Electrophoretic Mobility Shift Assay
For protein extraction from tissue samples the method of Negoro et al36 was used with modifications. Briefly, frozen arterial pieces were pulverized in a metallic chamber and resuspended in 1 mL cold extraction buffer containing 20 mmol/L HEPES-NaOH, pH 7.6, 20% (vol-vol) glycerol, 0.35 mol/L NaCl, 5 mmol/L MgCl2, 0.1 mmol/L EDTA, 1 mmol/L dithiothreitol (DTT), 0.5 mmol/L phenylmethyl sulfonyl fluoride (PMSF), and 1 µg/mL pepstatin A. The homogenate was vigorously shaken for 30 minutes, insoluble materials were precipitated by centrifugation at 40,000 x g for 30 minutes at 4°C, and the supernatant was dialyzed overnight against a binding buffer containing 20 mmol/L HEPES-NaOH, pH 7.6, 20% (vol/vol) glycerol, 0.1 mol/L NaCl, 5 mmol/L MgCl2, 0.1 mmol/L EDTA, 1 mmol/L DTT and 0.5 mmol/L PMSF. The dialysate was cleared by centrifugation at 10,000 x g for 15 minutes at 4°C, pooled according to the treatment groups, and frozen at -80°C in aliquots until use. Protein concentration was quantified by the BCA method (Pierce).
Gel shift assays were performed with a commercial kit according to the
instructions of the manufacturer (Promega). Briefly, NF-B
(5'-AGTTGAGGGGACTTTCCCAGGC-3') consensus oligonucleotide was
[32P]-end-labeled by incubation for 10 minutes at 37°C
with 10 U T4 polynucleotide kinase (Promega) in a reaction containing
10 µCi [
-32P]ATP (3000 Ci/mmol) (Amersham), 70
mmol/L Tris-HCl, 10 mmol/L MgCl2, and 5 mmol/L DTT. The
reaction was stopped by adding EDTA to a final concentration of 0.05
mol/L. Cellular protein (10 µg) was equilibrated for 10 minutes in a
binding buffer containing 4% glycerol, 1 mmol/L MgCl2, 0.5
mmol/L EDTA, 0.5 mmol/L DTT, 50 mmol/L NaCl, 10 mmol/L Tris-HCl, pH
7.5, and 50 mg/mL of poly(dI-dC) (Pharmacia LKB, Uppsala, Sweden).
Labeled probe (0.35 pmols) was added to the reaction and incubated for
20 minutes at room temperature. When competition assays were performed,
a 100-fold excess of the cold probe was added to this buffer 10 minutes
prior to the addition of the labeled probe. For supershift assays, 1
µg of anti-p50 and anti-p65 antibodies (Chemikon, Temecula, CA and
Santa Cruz Biotechnology, Santa Cruz, CA, respectively) were added and
incubated for 1 hour. Positive controls were done using Hela cell
nuclear extracts and negative controls were done setting the reaction
without nuclear extract. The reactions were stopped by addition of gel
loading buffer (250 mmol/L Tris-HCL, 0.2% bromophenol blue,
0.2% xylene cyanol and 40% glycerol) and run on a
nondenaturing, 4% acrylamide gel at 100 V at room temperature in
TBE. The gel was dried and exposed to X-ray film.
NF-B Determination by Southwestern Histochemistry
This method was developed to detect the distribution and
DNA-binding activity of NF-B in situ using a
digoxigenin-labeled double-stranded DNA probe with a specific NF-B
consensus sequence according to a previously described technique with
modifications.37
The synthetic sense
(5'-AGTTGAGGGGACTTTCCCAGGC-3') and antisense
(5'-GCCTGGGAAAGTCCCCTCAACT-3') probes (Genosys Biotechnology, London)
were annealed by heating at 80°C for 2 minutes. The probes were
labeled with digoxigenin (DIG oligonucleotide 3'-end labeling,
Boehringer Mannheim). Arterial sections were dewaxed, rehydrated,
incubated with 5 mmol/L levamisole for 30 minutes, and fixed with 0.2%
paraformaldehyde. They were then digested with 0.5% pepsin in 1 N HCl
for 30 minutes and washed with HEPES buffer (10 mmol/L HEPES, 40 mmol/L
NaCl, 10 mmol/L MgCl2, 1 mmol/L DTT, 1 mmol/L EDTA, 0.25%
BSA, pH 7.4). Sections were incubated with 0.1 mg/mL DNAse I during 30
minutes and washed once with HEPES buffer with 10 mmol/mL EDTA instead
of MgCl2. The DNA binding reaction was performed by
incubation with 100 pmol/mL of the labeled DNA probe overnight at
37°C. The sections were washed with HEPES buffer, washing buffer
(0.3% Tween 20 in 0.1 mol/L maleic acid, 0.15 mol/L NaCl, pH 7.5) and
blocking solution (0.1x SSC, 0.1% SDS diluted 1:10 in washing buffer)
for 1 hour. Preparations were incubated with an anti-digoxigenin
antibody conjugated with alkaline phosphatase (Boehringer) overnight at
4°C and then washed with 0.1 mol/L Tris-HCl, 0.1 mol/L NaCl, 50
mmol/L MgCl2, pH 9.5, and with washing buffer. Afterwards,
preparations were incubated with 0.4% NBT and 0.32% X-Phosphate for 1
hour. Reaction was stopped by washing with 10 mmol/L Tris, pH 8.0, and
1 mmol/L EDTA. Preparations were mounted with glycerol. Preparations
without probe were used as negative controls and a mutant NF-B probe
was used to test the specificity of the technique (sense:
5'-AGTTGAGGCTCCTTTCCCAGGC-3' and antisense:
5'-GCCTGGGAAAGGAGCCTCAACT-3'). For the identification of
the cell types showing NF-B activity in the lesions, simultaneous
detection of NF-B and macrophages or VSMC was done. Sections were
washed in PBS before mounting and immunohistochemistry was done
according to the above described technique.
A semiquantitative score ranging from 0 to 5 was assigned to each preparation.
Statistical Analysis
Results are expressed as the mean ± SD. Significance was established using GraphPAD InStat (GraphPAD Software). Student's t-test and Wilcoxon nonparametric test were used to compare the data. Differences were considered significant when P < 0.05.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Total serum cholesterol increased about 30 times over the basal values after 4 weeks of 2% cholesterol diet (1703 ± 536 versus 53 ± 37 mg/dl). Quinapril treatment did not induce any significant change compared to untreated animals.
At the moment of injury, after two days of quinapril consumption, there was a significant diminution in serum ACE activity (2.5 ± 1 x 10-2 versus 8 ± 3.6 x 10-2 U/mL; P < 0.03) and in arterial blood pressure (56 ± 16 versus 83 ± 13 mmHg; P < 0.0001) in the animals receiving quinapril. However, there was no significant difference between vascular ACE activity in quinapril-treated and untreated rabbits (1.2 ± 0.2 x 10-2, n = 5, versus 1.1 ± 0.4 x 10-2 U/mg; n = 4). At the time of death (28 days), serum ACE activity could not be quantified due to the turbidity caused by the high amount of lipids. Vascular ACE activity (determined in the aorta) was increased about fourfold in the arteries of the untreated rabbits compared to the initial values (4.8 ± 0.2 x 10-2 U/mg, n = 9), but it was significantly lower in quinapril-treated rabbits (2.14 ± 0.2 x 10-2 U/mg, n = 7; P = 0.023).
Maximal lesion size was reduced about 60% in the quinapril group compared to the untreated animals determined as intima/media ratio (1.01 ± 0.8 versus 0.39 ± 0.4) and as maximal stenosis (328,691 ± 263,104 versus 101,547 ± 109,977 µm2). However, the reduction did not reach statistical significance.
Effect of Quinapril Treatment on Expression of Chemokines and Presence of Macrophages in the Lesions
Healthy control animals did not show any stain for MCP-1 and IL-8
(Figure 1A)
. In contrast, untreated
animals presented a marked neointimal stain for MCP-1 and IL-8 that was
significantly reduced in the group treated with quinapril (185,642
± 169,784 versus 45,582 ± 68,963 µm2;
P < 0.05 and 13.7 ± 1.2 versus
8.6 ± 3.8 semiquantitative score; P < 0.04
respectively) (Figure 1A)
. The medial layers were also stained for
MCP-1 and IL-8; although there was a lower presence of both chemokines
in the animals treated with quinapril compared to the untreated (20%
and 25% respectively), the reduction was not statistically
significant.
|
, there were no
macrophages in the control arteries, while a certain number were
present in the neointima of the untreated rabbits. The treatment with
quinapril reduced by 80% the neointimal area occupied by these cells
(105,729 ± 196,696 µm2
versus
20,213 ± 52,957 µm2; P < 0.04).
Furthermore, in the quinapril-treated animals there was also a 50%
reduction in the medial area occupied by macrophages, although it did
not reach statistical significance. In Figure 1B
a bar graph depicts
the association between the neointimal areas occupied by MCP-1 and
macrophages and the semiquantitative score for IL-8. No staining was
observed in the negative controls included in each experiment (data not
shown).
MCP-1 and IL-8 mRNA expression was quantified in the femoral arteries
by semiquantitative RT-PCR (30 cycles of amplification) in a pool of
total RNA from each group. Control animals showed no detectable mRNA
expression of MCP-1 or IL-8 (Figure 2)
,
whereas untreated rabbits presented a marked arterial expression of
IL-8 (9.9 arbitrary units) and MCP-1 (4.8 arbitrary
units). Quinapril-treated rabbits showed a diminution in
arterial IL-8 expression (2.1 versus 9.9 a.u.) and
MCP-1 expression (2.4 versus 4.8 a.u.) compared to the
untreated animals.
|
As depicted in Figure 3A
, healthy
animals showed stain for collagen I in the medial layer. Collagen I
staining was found increased in the media of the injured arteries
compared to the healthy ones, but no difference was noted between the
untreated and treated groups (8.2 ± 4.8 versus 10
± 7; NS, semiquantitative score) (Figure 3, B and C)
. Collagen I was
also present in the neointima of both groups in similar amounts
(10.5 ± 5 versus 11.8 ± 7; NS) (Figure 3, B and C)
.
|
.
|
Because PDGF-B is one of the main mitogens involved in VSMC
proliferation and migration, we studied the effect of quinapril
treatment on PDGF-B mRNA expression by in situ
hybridization. Control animals showed no stain for PDGF-B although
there was a great expression of this factor in both the intima and
media of the untreated animals (10.2 ± 4 and 10.6 ± 3.3,
respectively) (Figure 5)
, coinciding with
the areas stained for macrophages. Quinapril-treated animals presented
a marked reduction in the expression of this cytokine in the intima
(5.5 ± 5.2, not significant) and in the media (4.8 ±
5, P < 0.03). As negative controls we used the labeled
sense probe, the treatment with RNAse, and the omission of the probe.
As expected, no staining was seen in these controls (not shown).
|
B Activity in the Lesions
The presence of NF-B in the lesions was evaluated by
Southwestern histochemistry, a technique that allows determination of
its in situ activation. We studied the activation of this
factor in a representative number of animals of each group. We found
NF-B activity in the neointima and media of four out of five animals
in the untreated group but in only two out of five in the
quinapril-treated group (3 ± 1 versus 1.2 ± 0.8
and 3.4 ± 1 versus 0.8 ± 0.5 respectively,
semiquantitative score).
Figure 6A
presents an example of a
control rabbit artery where no staining was detected (Figure 6A
1), an
untreated (Figure 6A
2), and a quinapril-treated rabbit (Figure 6A
3). A
graph indicating semiquantitative scores in the neointima and the media
for both groups is also shown. We have previously seen that cultured
monocytes and VSMC possess NF-B activity that increases in response
to inflammatory stimuli commonly overexpressed in the atherosclerotic
lesion.21
However, it is not known which of these cells is
responsible for the increased activity of this nuclear factor in
vivo. To identify which cellular types possessed NF-B activity
in the lesion, we performed Southwestern technique and
immunohistochemistry using specific antibodies for rabbit macrophages
and VSMC. Figure 6B
1 shows a double immunohistochemistry of an
untreated animal where the brown area refers to VSMC and the pink to
macrophages. This section corresponds to the lesion of the untreated
rabbit of Figure 5A
2 and it can be observed that NF-B activity is
present in the area occupied by both cell types. Figure 6B
2 shows a
double staining of the cap zone of the lesion for NF-B and VSMC. The
arrow points to a cell with the NF-B-activated nuclei in dark blue
and the cytoplasm in brown. Figure 6B
3 is a detail of the
macrophage-occupied zone also showing activated nucleus in blue (arrow)
and the cytoplasm of the macrophages in brown. We
found no difference between the two groups in the cellular distribution
of activity.
|
B Activity in the Uninjured Vessels
Because short-term hyperlipidemia alone does not cause the
formation of atherosclerotic plaques in rabbits but increases ACE
activity in the vascular wall (as shown in our model), we also studied
the activation of NF-B by gel shift assay in a pool of proteins
obtained from the uninjured arteries of the animals on high cholesterol
diet for 4 weeks. As can be seen in Figure 7
, NF-B activity was increased in the
arteries of the untreated group (lane 1) compared to the
quinapril-treated animals (lane 2), whereas no NF-B activity was
present in the control group that did not receive the atherogenic diet
(lane 3). We then characterized the activity induced by the diet in the
aortas of rabbits by supershift assay with the antibodies for the most
common subunits of NF-B, p50 and p65. Both subunits were present in
the activated NF-B of the untreated animals (lane 4) because the
anti-p50 antibody caused a diminution of the main band and the
appearance of a super-retarded one (arrow, lane 5) whereas the anti-p65
antibody induced almost the disappearance of the main complex (lane 6).
A densitometric analysis of the heterodimer band appears at the bottom
of the figure.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
B binding
site in their promoters. Moreover, ACE inhibition reduced arterial
NF-B activation in macrophages and VSMC, two key cells in the
pathogenesis of atherosclerosis. Although the role of vascular renin-angiotensin system activation in the development of atherosclerotic lesions is rather well-established,38 it is not clear whether the tissular RAS may also participate in the inflammatory process leading to plaque rupture and thrombosis. However, some data suggest a relationship. For example, it has been shown that the main sources of tissue ACE in human atherosclerotic plaques are areas with inflammatory cells, especially those with clustered macrophages.39 In addition, ACE inhibitors have been found to reduce the incidence of acute ischemic syndromes.26
The diminution of chemokine expression in the neointima of
quinapril-treated rabbits is probably due to the significant reduction
in the number of macrophages invading that area; these cells are a
source of and a target for PDGF-B, a potent monocyte chemotactic factor
that can also elicit the expression of tissue factor.40
VSMC are also targets for PDGF-B which can cause their proliferation
through the activation of NF-B41
and the expression of
MCP-1.42
In humans, PDGF-B seems to play a role in the
development of atherosclerotic plaques because in coronary arteries
after angioplasty it is found colocalized with the PDGF-ß
receptor.43
In several experimental
models44
the administration of neutralizing
anti-PDGF-B antibodies reduced the intimal thickening. In
addition, in VSMC this growth factor induces a stable increase in the
activity of low-density lipoprotein receptor-related
protein.45
Several studies have demonstrated the effectiveness of ACE inhibitors in preventing the neointima formation in vessel wall.46,47 In our model, we have noted a diminution in the neointima formation in quinapril-treated animals, though the reduction did not reach statistical significance. Higher doses of ACE inhibitors for the inhibition of neointima formation have been used in the above referenced studies, suggesting that the need for a higher dose may be due to the relative difficulty of inhibiting ACE tissue activity. Rakugi et al22 have inhibited vascular ACE approximately 60%, similar to the result found with our model and reduced neointima formation in an experimental model administering 50 mg/Kg/day to rats. However, we have shown an attenuation in the size of the plaque employing a more therapeutic dose of quinapril (1 mg/Kg/day). It is possible that higher doses of quinapril could have a major effect on the lesion size but, as is well known, rabbits are very sensitive to ACE inhibition and we had high mortality rates with doses higher than 1 mg/Kg/day (not shown).
The presence of NF-B in human atherosclerotic lesions in the nuclei
of macrophages, VSMC, and endothelial cells48
has been
recently demonstrated. We have seen that in our model both macrophages
and VSMC express activated NF-B and that quinapril treatment reduces
the activity equally in both cell types. We have previously shown that
Ang II elicits NF-B activation and MCP-1 expression in VSMC and
monocytic cells.20
Therefore, inhibition of Ang II
generation due to quinapril treatment could account for the reduction
of NF-B activation and of IL-8, MCP-1, and PDGF-B expression in the
atherosclerotic lesions. Because it has been shown that ACE inhibition
promotes nitric oxide production by inhibiting degradation of
bradykinin49
and PDGF-B seems to inhibit the induction of
nitric oxide synthase activity,50
one may speculate that an
increment in NO generation may play a role in the overall reduction of
inflammatory cells because NO seems to be involved in the
down-regulation of IL-8 and MCP-1 expression in endothelial
cells51,52
and adhesion molecules ICAM and VCAM-1 in
VSMC53
through the blockade of NF-B generation. It is
also noteworthy that NF-B is necessary for VSMC
proliferation19
and diminution of its activation could
therefore contribute to reduce the amount of VSMC in the lesion. This
effect can be potentiated by a lower migration of VSMC into the
neointima, because there are less chemokines and angiotensin II that
can induce them to migrate.54
As an overall consequence,
there is a diminution in the number of VSMC in the treated animals
that, in addition to a lower macrophage presence, may account for the
reduction in lesion size observed after treatment with ACE
inhibitors.25
Correlation between the susceptibility to development of fatty streaks
and the activation of vascular55
and hepatic NF-B in
response to an atherogenic diet56
has been demonstrated. In
our model we observed an increase in arterial NF-B activation in
response to the atherogenic diet and a reduction of the activation in
response to the treatment. Because we also observed that after 4 weeks
of high-cholesterol diet there was a four-fold increase in vascular ACE
activity, one may also speculate that the lower NF-B activity
observed in the uninjured aortas of the quinapril-treated group could
be due to reduction of Angiotensin II generated by the
hyperlipidemia.57
Collagen I is the main extracellular matrix protein in the fibrous cap
that, together with collagen III, accounts for around 60% of the total
protein and at least 90% of the total collagen of the
lesion.58
The integrity of the fibrous cap and thus its
resistance to rupture depends critically on the collagenous
extracellular matrix of the plaque's fibrous cap. Collagen I is
produced mainly by VSMC and a diminution in collagen synthesis due to a
decreased number of VSMC, as well as an increment in its degradation
due to metalloproteinases secreted by the invading macrophages, have
been associated with the weakening of the fibrous cap making it prone
to rupture.59
Angiotensin II may increase plaque stability
by favoring collagen I expression, an effect that could theoretically
be reversed by ACE inhibition. Paradoxically, the treatment with
quinapril did not modify the expression of collagen I. The reasons for
this apparent discrepancy are unknown but one possible explanation
could be that whereas MCP-1, IL-8, and PDGF-B are NF-B-dependent
genes, the expression of collagen I is regulated by the transcription
factor AP-1.60
This suggests that the blockade of NF-B
activation as a potential control step of certain genes may be achieved
with quinapril treatment, although angiotensin II can also regulate
gene expression via AP-1 activation.61
Further
studies are needed to clarify this issue. Consequently, untreated and
quinapril-treated animals showed plaques with fibrotic morphology, but
only those treated with quinapril presented a diminution of the
inflammatory components IL-8, MCP-1, PDGF-B, and macrophages.
However, it is also possible that some of the effects observed with the ACE inhibitor quinapril could be due in part to the accumulation of bradykinins or the generation of NO and prostaglandins. In fact, although previous authors have shown that other ACE inhibitors or AT1 antagonists could cause diminution of the atherosclerosis lesion,62 the potential mechanisms of this beneficial effect are mostly unknown. To establish the relative roles of Angiotensin II and bradykinin in the actions of quinapril, similar studies with bradykinin or AT1 antagonist may be necessary; the aim of our study was not to compare the effect of these drugs.
On the whole, our results support the conclusion that in a model of atherosclerosis in rabbits, ACE inhibition improves some inflammatory and proliferative parameters of the lesion without affecting the fibrotic component and could therefore contribute to stabilization of the plaque.
|
Acknowledgements |
|---|
|
Footnotes |
|---|
Supported by grants from Ministerio de Educación y Ciencia (MEC) (PB 94/0211; SAF 97-55 PM97/85), Fundación Mapfre Medicina and Parke-Davis, Spain.
Accepted for publication July 24, 1998.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
B: a pivotal transcription factor in chronic inflammatory diseases. N Engl J Med 1997, 336:1066-1071B and SP1 regulate transcription of the human monocyte chemoattractant protein 1 gene. J Immunol 1994, 153:2052-2063[Abstract]
B interacts functionally with the platelet-derived growth factor B-chain shear-stress response element in vascular endothelial cells exposed to fluid shear stress. J Clin Invest 1995, 96:1169-1175
B-like activity is essential for proliferation of cultured bovine vascular smooth muscle cells. J Clin Invest 1995, 96:2521-2527
B activation, monocyte chemoattractant protein-1 expression, and macrophage infiltration in a rabbit model of early accelerated atherosclerosis. Circulation 1997, 95:1532-1541. The role of constitutive NF-B activity. J Biol Chem 1996, 271:11317-11324This article has been cited by other articles:
 |
|
 |
|
  A. J. Flammer, I. Sudano, F. Hermann, S. Gay, A. Forster, M. Neidhart, P. Kunzler, F. Enseleit, D. Periat, M. Hermann, et al. Angiotensin-Converting Enzyme Inhibition Improves Vascular Function in Rheumatoid Arthritis Circulation, April 29, 2008; 117(17): 2262 - 2269. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Bro, C. J. Binder, J. L. Witztum, K. Olgaard, and L. B. Nielsen Inhibition of the Renin-Angiotensin System Abolishes the Proatherogenic Effect of Uremia in Apolipoprotein E-Deficient Mice Arterioscler. Thromb. Vasc. Biol., May 1, 2007; 27(5): 1080 - 1086. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Tedgui and Z. Mallat Cytokines in Atherosclerosis: Pathogenic and Regulatory Pathways Physiol Rev, April 1, 2006; 86(2): 515 - 581. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Altunoluk, H. Soylemez, F. Oguz, E. Turkmen, and E. Fadillioglu An Angiotensin-Converting Enzyme Inhibitor, Zofenopril, Prevents Renal Ischemia/Reperfusion Injury in Rats Ann. Clin. Lab. Sci., January 1, 2006; 36(3): 326 - 332. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 O Wazni, D O Martin, N F Marrouche, M Shaaraoui, M K Chung, S Almahameed, R A Schweikert, W I Saliba, and A Natale C reactive protein concentration and recurrence of atrial fibrillation after electrical cardioversion Heart, October 1, 2005; 91(10): 1303 - 1305. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. J.E. Sattler, J. E. Woodrum, O. Galili, M. Olson, S. Samee, F. B. Meyer, X.-Y. Zhu, L. O. Lerman, and A. Lerman Concurrent Treatment With Renin-Angiotensin System Blockers and Acetylsalicylic Acid Reduces Nuclear Factor {kappa}B Activation and C-Reactive Protein Expression in Human Carotid Artery Plaques Stroke, January 1, 2005; 36(1): 14 - 20. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Deo, A. Khera, D. K. McGuire, S. A. Murphy, J. de P. Meo Neto, D. A. Morrow, and J. A. de Lemos Association among plasma levels of monocyte chemoattractant protein-1, traditional cardiovascular risk factors, and subclinical atherosclerosis J. Am. Coll. Cardiol., November 2, 2004; 44(9): 1812 - 1818. [Abstract] [Full Text] [PDF]
|
|
